Plant alpha farnesene synthase and polynucleotides encoding same

ABSTRACT

The present invention provides an isolated alpha-farnesene synthase and polynucleotide sequences encoding the enzyme. The invention also provides nucleic acid constructs, vectors and host cells incorporating the polynucleotide sequences. It further relates to the production of alpha-farnesene using the enzyme and modulation of alpha-farnesene synthesis in plants and selection of plants with altered alpha-farnesene synthase activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT InternationalApplication No. PCT/NZ2003/000229, filed on Oct. 15, 2003, which claimsbenefit of NZ 521984, filed on Oct. 15, 2002, which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to the enzyme alpha-farnesene synthase andto polynucleotide sequences encoding the enzyme. The invention alsorelates to nucleic acid constructs, vectors and host cells incorporatingthe polynucleotide sequences. It further relates to the production ofalpha-farnesene and its use in products such as an insect attractant, asex pheromone and other products. Alpha-farnesene may also be used toproduce other products with characteristic aromas useful as flavours andfragrances.

BACKGROUND ART

Alpha-farnesene (FIG. 1) is an acyclic sesquiterpene hydrocarbon(C₁₅H₂₄; 3,7,11-trimethyl-1,3,6,10-dodecatetraene) that is eitherconstitutively present or induced in a wide range of species.

The biosynthetic pathway for the sesquiterpenes branches off from thegeneral terpenoid pathway, beginning with the allylic diphosphate esterfarnesyl diphosphate (FDP, also shortened to FPP) (Bohlmann, et al.,Proc. Natl. Acad. Sci. U.S.A. 95, 4126-4133 (1998), Cane and Bowser,Bioorg. Med. Chem. Lett. 9, 1127-1132 (1999), Davis and Croteau, Top.Curr. Chem. 209, 53-95 (2000)). Alpha-farnesene is synthesised from FDPin a reaction that proceeds through a carbocation intermediate (FIG. 2)and is catalysed by the sesquiterpene synthase alpha-farnesene synthase(Rupasinghe, et al., J. Am. Soc. Hortic. Sci. 123, 882-886 (1998)). Thepathway for sesquiterpene biosynthesis, the acetate/mevalonate pathway,is localised to the cytoplasm; in contrast to the pathways formonoterpene and diterpene biosynthesis, which occur in the chloroplast(Croteau, et al., In Biochemistry and Molecular Biology of Plants, edsBuchanan, Gruissem and Jones, American Society of Plant Physiologists,1250-1318 (2000); Lange, et al., Proc. Natl. Acad. Sci. U.S.A. 97,13172-13177 (2000)).

All known plant terpene synthases, however, whether monoterpene,sesquiterpene or diterpene, appear to be closely related. Similaritiesinclude the positioning of intron sequences (Trapp and Croteau, Genetics158, 811-832 (2001)) and the presence of conserved sequences, such as anaspartate-rich DDXX(D,E) motif (Lesburg, et al., Curr. Opin. Struct.Biol. 8, 695-703 (1998)). This motif is involved in the binding of metalions, usually Mg²⁺, that are necessary for catalysis. (Lesburg, et al.,Curr. Opin. Struct. Biol. 8, 695-703 (1998)).

Alpha-farnesene synthase has been partially purified from the skin ofapple fruit (Malus domestica Delicious). However, poor recovery andinstability of the partially purified enzyme restricted furtherpurification (Rupasinghe, et al., J. Am. Soc. Hortic. 125, 111-119(2000)).

Alpha-farnesene is an insect attractant. It is a sex pheromone in miceand insects. Oxygenated (including chemicals occurring on exposure toair) alpha-farnesene products (eg farnesol, farnesal) havecharacteristic aromas (flavour/fragrance use). Other uses foralpha-farnesene and its derivatives are as potent cancer preventionagents, and in plastic film synthesis.

There is also a link between both the levels of alpha-farnesene and itsoxidation products and the development of superficial scald, apostharvest physiological disorder that appears as a dark coloration ofthe apple skin following cool storage (Watkins, et al., Acta Hort. 343,155-160 (1993), Ju and Bramlage, J. Am. Soc. Hortic. Sci. 125, 498-504(2000), Whitaker and Saftner, J. Agric. Food Chem. 48, 2040-2043 (2000),Rowan, et al., J. Agric. Food Chem. 49, 2780-2787 (2001)). To date thecausal relationship between alpha-farnesene and scald is still unclear(Ju and Curry, J. Am. Soc. Hortic. Sci. 125, 626-629 (2000), Rupasinghe,et al., J. Am. Soc. Hortic. Sci. 125, 111-119 (2000)). Ethyleneproduction and alpha-farnesene biosynthesis also appear to be closelyassociated (Watkins, et al., Acta Hort. 343, 155-160 (1993), Fan, etal., J. Agric. Food Chem. 47, 3063-3068 (1999)). Recently it has beenshown that ethylene may regulate the biosynthesis of alpha-farneseneduring fruit ripening by acting on the mevalonate pathway, specificallyby inducing the conversion of hydroxymethylglutaryl CoA to mevalonicacid (Ju and Curry, J. Am. Soc. Hortic. Sci. 125, 105-110 (2000), Ju andCurry, Postharvest Biol. Technol. 19, 9-16 (2000), Ju and Curry, J. Am.Soc. Hortic. Sci. 126, 491-495 (2001)).

It is an object of the invention to provide methods for in vitrosynthesis of alpha-farnesene and/or for genetically modifying plants toalter the levels of alpha-farnesene synthase activities in plants;and/or to offer the public a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the invention provides an isolated polynucleotideencoding alpha-farnesene synthase. In a preferred embodiment thepolynucleotide encodes a polypeptide comprising at least one repeat ofDDXXD and (L,V)(V,L,A)(N,D)(L,I,V)X(S,T)XXXE, wherein X is any aminoacid.

In a further aspect the invention provides an isolated polynucleotide ofSEQ ID NO:1 also (shown in FIG. 3) or a fragment or variant thereofwherein the fragment or variant encodes a polypeptide withalpha-farnesene synthase activity.

In a further aspect, the invention provides an isolated polynucleotideencoding the polypeptide of SEQ ID NO:2 (shown in FIG. 4) or encoding avariant or a fragment of that sequence which has alpha-farnesenesynthase activity.

In a further aspect the invention provides an isolated alpha-farnesenesynthase polypeptide.

In yet a further aspect, the invention provides an isolatedalpha-farnesene synthase having the sequence SEQ ID NO:2 or a fragmentor variant thereof with alpha-farnesene synthase activity.

The polypeptides of the invention are useful for in vitro preparation ofalpha-farnesene.

In a further aspect the invention provides a genetic constructcomprising a polynucleotide of the invention.

In yet a further aspect the invention provides a genetic constructcomprising in the 5′-3′ direction

an open reading frame polynucleotide encoding a polypeptide of theinvention.

Preferably the genetic construct also comprises a promoter sequence.

Preferably the genetic construct further comprises a terminationsequence.

In another aspect the invention provides a genetic construct comprisingin the 5′-3′ direction a polynucleotide which hybridizes to apolynucleotide encoding a polypeptide of the invention.

Preferably the genetic construct also comprises a promoter sequence.

Preferably the genetic construct further comprises a terminationsequence.

In a further aspect the invention provides a vector comprising a geneticconstruct of the invention.

In a further aspect the invention provides a host cell comprising agenetic construct of the invention.

In still a further aspect, the invention provides a transgenic plantcell which includes a genetic construct of the invention.

In addition the invention provides a transgenic plant comprising suchcells.

In another aspect the invention provides a method for preparingalpha-farnesene comprising the steps of

-   (a) culturing a cell which has been genetically modified with a    polynucleotide of the invention to provide increased alpha-farnesene    synthase activity;-   (b) providing the cell with farnesyl diphosphate if necessary; and-   (c) separating the alpha-farnesene produced.

This method of the invention allows use of biofermentation for aconvenient method for preparing the product.

In a further aspect the invention provides a method for preparingalpha-farnesene comprising the steps of

-   -   (a) obtaining a polypeptide of the invention    -   (b) incubating farnesyl diphosphate in the presence of the        polypeptide and    -   (c) separating the alpha-farnesene produced.

In a further aspect the invention comprises a method for modulating thealpha-farnesene production of a plant, the method comprising: increasingor decreasing expression of alpha-farnesene synthase wherein saidincreasing or decreasing is achieved by genetic modification to alterthe expression of a gene encoding an alpha-farnesene synthase. Themodified cell and plants comprising such a cell also form part of theinvention.

In a further aspect the invention there is provided a polynucleotidecomprising at least 15 contiguous nucleotides from SEQ ID NO: 1

In a further aspect the invention comprises a method of selecting aplant with altered alpha-farnesene content comprising the steps of:

-   (a) contacting polynucleotides from at least one plant with at least    one polynucleotide comprising at least 15 contiguous nucleotides of    the polynucleotide of claim 1 to assess the expression of    alpha-farnesene synthase; and-   (b) selecting a plant showing altered expression.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be better understood with reference to theaccompanying drawings in which:

FIG. 1 shows the structures of the isomers of alpha-farnesene.

FIG. 2 shows the pathway for alpha-farnesene synthesis in apple.

FIG. 3 shows the cDNA sequence that encodes alpha-farnesene synthase.The sequence was obtained from a cDNA library that was constructed fromRoyal Gala 150 days after full bloom (DAFB) apple skin.

FIG. 4 shows the predicted amino acid sequence of alpha-farnesenesynthase from apple skins. The DDXXD motif involved in the binding ofthe metal ions necessary for catalysis is in bold. The highly conservedconsensus sequence (L,V)(V,L,A)(N,D)D(L,I,V)X(S,T)XXXE, also involved inmetal ion binding, is underlined.

FIG. 5 shows a GC-MS trace of headspace above Royal Gala apples showing(E,E) alpha-farnesene peak at retention time 42.57 minutes.

FIG. 6 shows a GC-MS trace of headspace above nickel purified cell freeextracts (in binding buffer) harbouring alpha-farnesene synthase cDNAshowing (E,E) alpha-farnesene (retention time 43.09 minutes) and (Z,E)alpha-farnesene (retention time 42.29 minutes).

FIG. 7 shows isomeric forms of alpha-farnesene produced in vitro bypurified recombinant alpha-farnesene synthase in response to feeding aprecursor FDP of mixed isomers.

FIG. 8 shows the protein elution profile and approximate molecular massof alpha-farnesene synthase on Sephacryl S-300 HR Gel filtrationchromatography.

FIG. 9 shows alpha-farnesene synthase activity of fractions of purifiedprotein after application to S-Sephacryl 300 HR Gel Filtration.

FIG. 10 shows the optimum pH for alpha-farnesene synthase activity. Dataare means plus SE of means based on 3 replicates per experiment. Assayconditions include saturating Mg⁺⁺/Mn⁺⁺ (7 mM/150 μM) at pH 7.5.

FIG. 11 shows effect of increasing concentrations of FDP onalpha-farnesene synthase activity in the presence of saturating metalions. Assay conditions include saturating FDP (25 μM) and saturatingMg⁺⁺/Mn⁺⁺ (7 mM/150 μM).

FIG. 12 shows effect of Mg²⁺ on activity of alpha-farnesene synthasewith saturating FDP (25 μM).

FIG. 13 shows effect of Mn²⁺ on activity of alpha-farnesene synthasewith saturating FDP (25 μM). Data are means and SE of three replicatesfrom 1 experiment.

FIG. 14 shows the activity of alpha-farnesene synthase at differenttemperatures measured with saturating FDP (25 μM) and metal cofactorsMg⁺⁺/Mn⁺⁺ (7 mM/150 μM).

FIG. 15 shows an example of one experiment showing production of E,Ealpha-farnesene in N. benthamiana leaves, with and without infiltrationwith FDP.

FIG. 16 shows PCR amplification of genomic DNA extracted from transgenicArabidopsis thaliana plants using primers designed from the 5′ end ofthe alpha-farnesene synthase cDNA sequence. The size of the expectedamplification product is 513 bp. The lane labelled MW representsstandard molecular weight markers (Invitrogen). Lanes labelled 1, 2, 3and 4 are independent transgenic Arabidopsis thaliana lines containingthe alpha-farnesene synthase cDNA insert. The lane labelled apple showsthe amplification product resulting from RT-PCR of total RNA extractedfrom ‘Royal Gala’ apple peel.

FIG. 17 shows Southern analysis of a BamH1 digest of genomic DNAextracted from transgenic Arabidopsis thaliana plants using as a probean 810 base pair 32P-labelled PCR fragment that was amplified from EST57400 with the primers 57400_A3 (5′ AGAGTTCACTTGCAAGCTGA 3′ (SEQ IDNO:3)) and 57400NR1 (5′ GGATGCTTCCCT 3′ (SEQ ID NO:4)). The size of theBamH1 restriction fragment containing cDNA for alpha-farnesene synthaseis 2050 base pairs. The lane labelled MW is the molecular weight marker(Invitrogen). pHEX is refers to genomic DNA extracted from transgenicArabidopsis thaliana plants containing the transformation vector only,without the alpha-farnesene synthase cDNA insert. Lanes labelled 1, 2, 3and 4 are independent transgenic Arabidopsis thaliana lines containingthe alpha-farnesene synthase cDNA insert. kb=kilobases

FIG. 18 shows PCR amplification of total RNA extracted from seedlings,leaves and flowers of transgenic Arabidopsis thaliana Line 3 plantsusing primers designed from the 5′ end of the alpha-farnesene synthasecDNA sequence (a); from internal sequences (b) and from near the 3′ endof the α-farnesene synthase cDNA sequence (c). The size of theamplification product expected for (a) is 513 bp, for (b) is 349 bp andfor (c) is 180 bp. The lane labelled MW is the molecular weight marker(Invitrogen). Lanes labelled 1 contain the resulting products from PCRamplification of the total RNA and Lanes labelled 2 contain theresulting products from RT-PCR amplification of the total RNA. bp=basepairs

FIG. 19 shows headspace volatiles present in A. thaliana inflorescencesfrom Line 3 plants expressing alpha-farnesene synthase gene and fromcontrol plants expressing empty vector.

FIG. 20 shows Northern analysis of total RNA extracted from varioustissues of Malus domestica using as a probe a 350 base pair DIG-labelledPCR fragment that was amplified from EST 57400 with the primers 57400NF1(5′ GCACATTAGAGAACCACCAT 3′ (SEQ ID NO:5)) and 57400NR1 (5′ GGATGCTTCCCT3′ (SEQ ID NO:4)). DAFB=days after full bloom.

-   A. A histogram of alpha-farnesene synthase mRNA levels that were    present in the total RNA extracted from each tissue.-   B. A histogram of alpha-farnesene synthase mRNA levels that were    present in the total RNA extracted from tissues that are expressing    alpha-farnesene synthase at lower levels than in ‘Royal Gala’ 150    DAFB fruit peel or ‘Aotea’ expanding leaf-   C. i Northern analysis of alpha-farnesene synthase mRNA.    -   ii Hybridisation of the 18S ribosomal RNA.

FIGS. 21 a and 21 b show a phylogenetic analysis of terpene synthases ofknown function and shows that alpha-farnesene synthase forms a uniquelade.

DETAILED DESCRIPTION

In one embodiment of the invention, cells genetically modified toexhibit alpha-farnesene synthase activity are used for the production ofalpha-farnesene. While the cells may potentially be of any cell typethat can be grown in culture, it is currently preferred to use bacteriaor yeast cells for producing alpha-farnesene (and its oxidation productsor derivatives). Preferred cells for use in the biofermentationprocesses of this embodiment are cells with GRAS status, for exampleappropriate E. coli strains, Lactobacillus sp and other non-pathogenicGRAS status bacteria or yeasts such as brewers yeast.

Alpha-farnesene (or derivatives of alpha-farnesene) produced bybiofermentation may be used as pheromones for use in insect or rodentcontrol; as flavour or fragrance additives to food, medicine,toothpastes or perfumes; for the manufacture of pharmaceuticals withanti-tumour, anti-candida, mucosal stabilizing, anti-inflammatory andanti-ulcerative properties, for the manufacture of films and polymersfor use in packaging and moulded articles, particularly degradableplastics, general agrochemical production, production of solvents forindustrial cleaning (eg algaecides) and membranes for dewaxing solventsor oils.

In an alternative to alpha-farnesene production by biofermentation,alpha-farnesene synthase may be extracted and optionally immobilised andused in alpha-farnesene production. For example cultured cells asdescribed above may be used as the source of alpha-farnesene synthase.The enzyme may for example be immobilised on beads, for example alginatebeads.

In another aspect of the invention, the polynucleotides of the inventionare used to prepare transgenic plants that over-express thealpha-farnesene synthase in at least some parts of the plant. In thisway the invention is used to impart fragrance to flowers, to repel orattract insects (either as indicator plants, host plants, or alternativehosts) or to impart an altered flavour to fruit or vegetables or toprevent scald in fruit, or to extract pharmaceutical products or animalor insect efficacious extracts.

In one particular aspect the polynucleotides of the invention are usedin plants of the order Rosaceae, particularly in the genus Malus toprovide increased flavour in fruit.

In another aspect polynucleotides of the invention are used to decreasealpha-farnesene synthase activity in apple fruit. This may be achievedin several ways, for example by genetically modifying the apples so thatalpha-farnesene synthase polynucleotide is transcribed in an antisenseorientation which results in decreased alpha-farnesene synthasetranslation. Such fruit may then display decreased superficial scald inapple skin following cold storage or be less attractive to insects suchas the codling moth.

In another aspect the invention provides a method useful in applebreeding. Segments of the polynucleotide sequences of the invention maybe used as probes or primers to investigate the genetic makeup ofcandidate apple varieties with respect to alpha-farnesene synthaseactivity. The presence of high levels of polynucleotides encodingalpha-farnesene synthase activity in the fruit of apples may be used toidentify apples with added flavour and presence of low levels may beused to identify apples with favourable storage properties or insectresistance.

The amino acid sequence of one polypeptide, an alpha-farnesene synthasefrom apple, and that of the polynucleotide sequence encoding it aregiven in FIGS. 4 and 3 respectively (SEQ ID NO:2 and SEQ ID NO:1). Itwill however be appreciated that the invention is not restricted only tothe polynucleotide/polypeptide having the specific nucleotide/amino acidsequence given in FIGS. 3 and 4. Instead, the invention also extends tovariants of the polynucleotide/polypeptide of FIGS. 3 and 4 which encodeor possess alpha farnesene synthase activity.

The term “polynucleotide(s)” as used herein means a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesand includes DNA and corresponding RNA molecules, including hnRNA andmRNA molecules, both sense and anti-sense strands, and comprehends cDNA,genomic DNA and recombinant DNA, as well as wholly or partiallysynthesized polynucleotides. An hnRNA molecule contains introns andcorresponds to a DNA molecule in a generally one-to-one manner. An mRNAmolecule corresponds to an hnRNA and DNA molecule from which the intronshave been excised. A polynucleotide may consist of an entire gene, orany portion thereof. Operable anti-sense polynucleotides may comprise afragment of the corresponding polynucleotide, and the definition of“polynucleotide” therefore includes all such operable anti-sensefragments.

The term ‘polypeptide(s)’ as used herein includes peptides, polypeptidesand proteins.

The phrase “variants with alpha-farnesene synthase activity” is used inrecognition that it is possible to vary the amino acid/nucleotidesequence of a polypeptide/polynucleotide while retaining substantiallyequivalent functionality. The equivalent can be, for example, a fragmentof the polypeptide, a fusion of the polypeptide with another polypeptideor carrier, or a fusion of a fragment with additional amino acids.

An “isolated” polypeptide is a polypeptide that has been identified andseparated or recovered to be largely free of components of its naturalenvironment, (that is so that the polypeptide comprises at least 50% ofthe polypeptides from its natural environment, preferably at least 80%,more preferably at least 90%). The term “isolated” polypeptide includespolypeptides in situ within recombinant cells. However generallyisolated polypeptides will be prepared by at least one purificationstep.

An “isolated” polynucleotide is a nucleotide molecule that is identifiedand separated from at least one contaminant polynucleotide with which itis ordinarily associated.

Variant polynucleotide sequences also include equivalent sequences,which vary in size, composition, position and number of introns, as wellas size and composition of untranslated terminal regions. Variantpolynucleotides also include those encoding functionally equivalentpolypeptides.

It will be understood that a variety of substitutions of amino acids ispossible while preserving the structure responsible for activity of thepolypeptides. Conservative substitutions are described in the patentliterature, as for example, in U.S. Pat. No. 5,264,558 or U.S. Pat. No.5,487,983. It is thus expected, for example, that interchange amongnon-polar aliphatic neutral amino acids, glycine, alanine, proline,valine and isoleucine, would be possible. Likewise, substitutions amongthe polar aliphatic neutral amino acids, serine, threonine, methionine,asparagine and glutamine could be made. Substitutions among the chargedacidic amino acids, aspartic acid and glutamic acid, could probably bemade, as could substitutions among the charged basic amino acids, lysineand arginine. Substitutions among the aromatic amino acids, includingphenylalanine, histidine, tryptophan and tyrosine are also possible.Such substitutions and interchanges are well known to those skilled inthe art.

Equally, nucleotide sequences encoding a particular product can varysignificantly simply due to the degeneracy of the nucleic acid code.

A polynucleotide or polypeptide sequence may be aligned, and thepercentage of identical nucleotides in a specified region may bedetermined against another sequence, using computer algorithms that arepublicly available. Two exemplary algorithms for aligning andidentifying the similarity of polynucleotide sequences are the BLASTNand FASTA algorithms. The similarity of polypeptide sequences may beexamined using the BLASTP algorithm. Both the BLASTN and BLASTP softwareare available on the NCBI anonymous FPT server. The BLASTN algorithmversion 2.0.4 [Feb.-24-1998], set to the default parameters described inthe documentation of variants according to the present invention. Theuse of the BLAST family of algorithms, including BLASTN and BLASTP, isdescribed at NCBI's website at URL and in the publication of Altschul etal., Nucleic Acids Res. 25, 3389-34023 (1997). The computer algorithmFASTA is available on the Internet. Version 2.0u4, February 1996, set tothe default parameters described in the documentation and distributedwith the algorithm, is also preferred for use in the determination ofvariants according to the present invention. The use of the FASTAalgorithm is described in Pearson and Lipman Proc. Natl. Acad. Sci. USA85, 2444-2448 (1988), Pearson Methods in Enzymology 183, 63-98 (1990).

The following running parameters are preferred for determination ofalignments and similarities using BLASTN that contribute to E values (asdiscussed below) and percentage identity: Unix running command: blastall-p blastn -d embldb -e 10 -G 1 -E 1 -r 2 -v 50 -b 50 -I queryseq -oresults; and parameter default values:

-   -p Program Name [String]-   -d Database [String]-   -e Expectation value (E) [Real]-   -G Cost to open a gap (zero invokes default behaviour) [Integer]-   -E Cost to extend a cap (zero invokes default behaviour) [Integer]-   -r Reward for a nucleotide match (blastn only) [Integer]-   -v Number of one-line descriptions (V) [Integer]-   -b Number of alignments to show (B) [Integer]-   -i Query File [File In]-   -o BLAST report Output File [File Out] Optional

For BLASTP the following running parameters are preferred: blastall -pblastp -d swissprotdb -e 10 -G 1 -E 1 -v 50 -b 50 -I queryseq -o results

-   -p Program Name [String]-   -d Database [String]-   -e Expectation value (E) [Real]-   -G Cost to open a gap (zero invokes default behaviour) [Integer]-   -E Cost to extend a cap (zero invokes default behaviour) [Integer]-   -v Number of one-line descriptions (v) [Integer]-   -b Number of alignments to show (b) [Integer]-   -i Query File [File In]-   -o BLAST report Output File [File Out] Optional

The “hits” to one or more database sequences by a queried sequenceproduced by BLASTN, BLASTP, FASTA, or a similar algorithm, align andidentify similar portions of sequences. The hits are arranged in orderof the degree of similarity and the length of sequence overlap. Hits toa database sequence generally represent an overlap over only a fractionof the sequence length of the queried sequence.

The BLASTN and FASTA algorithms also produce “Expect” or E values foralignments. The E value indicates the number of hits one can “expect” tosee over a certain number of contiguous sequences by chance whensearching a database of a certain size. The Expect value is used as asignificance threshold for determining whether the hit to a database,such as the preferred EMBL database, indicates true similarity. Forexample, an E value of 0.1 assigned to a hit is interpreted as meaningthat in a database of the size of the EMBL database, one might expect tosee 0.1 matches over the aligned portion of the sequence with a similarscore simply by chance. By this criterion, the aligned and matchedportions of the sequences then have a 90% probability of being the same.For sequences having an E value of 0.01 or less over aligned and matchedportions, the probability of finding a match by chance in the EMBLdatabase is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, “variant” polynucleotides, with referenceto each of the polynucleotides of the present invention, preferablycomprise sequences having the same number or fewer nucleic acids thaneach of the polynucleotides of the present invention and producing an Evalue of 0.01 or less when compared to the polynucleotide of the presentinvention. That is, a variant polynucleotide is any sequence that has atleast a 99% probability of being the same as the polynucleotide of thepresent invention, measured as having an E value of 0.01 or less usingthe BLASTN or FASTA algorithms set at the parameters discussed above.

Variant polynucleotide sequences will generally hybridize to the recitedpolynucleotide sequence under stringent conditions. As used herein,“stringent conditions” refers to prewashing in a solution of 6×SSC, 0.2%SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by twowashes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of30 minutes each in 0.2×SSC, 0.1% SDS at 65° C. The variantpolynucleotide sequences of the invention are at least 50 nucleotides inlength.

Variant polynucleotides also include sequences which have a sequenceidentity of at least 25% or at least 60%, generally 70%, preferably 80%,more preferably 90%, even more preferably 95%, very preferably 98% andmost preferably 99% or more to the nucleotide sequence given in thesequence listing herein.

In general, polypeptide sequences that code for the alpha-farnesenesynthases of the invention will be at least 25% or at least 50%,generally at least 60%, preferably 70%, and even 80%, 85%, 90%, 95%,98%, most preferably 99% homologous or more with the disclosed aminoacid sequence. That is, the sequence similarity may range from 25% to99% or more. In addition the invention includes polynucleotide sequencesencoding these amino acid sequences.

Also encompassed by the invention are fragments of the polynucleotideand polypeptide sequences of the invention. Polynucleotide fragments mayencode protein fragments which retain the biological activity of thenative protein. Alternatively, fragments used as hybridisation probesgenerally do not encode biologically active sequences. Fragments of apolynucleotide may range from at least 15, 20, 30, 50, 100, 200, 400 or1000 contiguous nucleotides up to the full length of the nativepolynucleotide sequences disclosed herein.

Fragments of the polypeptides of the invention will comprise at least 5,10, 15, 30, 50, 75, 100, 150, 200, 400 or 500 contiguous amino acids, orup to the total number of amino acids in the full length polypeptides ofthe invention.

Variant is also intended to allow for rearrangement, shifting orswapping of one or more nucleotides or domains/motifs (from coding,non-coding or intron regions) from genes (including terpene synthases)from the same or other species, where such variants still provide afunctionally equivalent protein or polypeptide of the invention orfragment thereof.

It is, of course, expressly contemplated that homologs to thespecifically described alpha-farnesene synthase having the sequence ofFIG. 4 (SEQ ID NO:2) exist in other plants. Such homologs are also“variants” as the phrase is used herein.

A polynucleotide sequence of the invention may further comprise one ormore additional sequences encoding one or more additional polypeptides,or fragments thereof, so as to encode a fusion protein. Systems for suchrecombinant expression include, but are not limited to, mammalian,bacteria and insect expression systems. Also contemplated are cell-freeexpression systems.

DNA sequences from plants other than Malus domestica which are homologsof the alpha-farnesene synthase of FIG. 3 (SEQ ID NO:1) may beidentified (for example by computer-aided searching of private or publicand sequence databases. Alternatively, probes based on the sequence ofFIG. 4 can be synthesized and used to identify positive clones in eithercDNA or genomic DNA libraries derived from other plants by means ofhybridization methods. PCR-based techniques includingreverse-transcriptase (RT)-PCR may also be employed. Probes and/or PCRprimers should be at least about 10, preferably at least about 15 andmost preferably at least about 20 nucleotides in length. Hybridizationand PCR techniques suitable for use with such oligonucleotide probes arewell known in the art. Positive library clones or PCR products may beanalyzed by restriction enzyme digestion, DNA sequencing or the like.

The polynucleotides of the present invention may be generated bysynthetic means using techniques well known in the art. Equipment forautomated synthesis of oligonucleotides is commercially available fromsuppliers such as Perkin Elmer/Applied Biosystems Division (Foster City,Calif.) and may be operated according to the manufacturer'sinstructions.

Allelic variation in Malus domestica has been observed. Thealpha-farnesene synthase polypeptide of the variety Aotea differs fromthat of SEQ ID NO:2 by 5 amino acids over a partial sequence. Partialpolynucleotide sequences for the alpha-farnesene synthase gene in Aoteaand the corresponding polypeptide are those of SEQ ID NO:6 and SEQ IDNO: 7 respectively.

(SEQ ID NO:6) CTAAGTTGGAGCTCATTGACAGCGTCCGAAAACTAGGCCTCGCGAACCTCTTCGAAAAGGAAATCAAGGAAGCCCTAGACAGCGTTGCAGCTATCGAAAGCGACAATCTCGGCACAAGAGACGATCTCTATGCTACTGCATTACACTTCAAGATCCTCAGGCAGCATGGCTATAAAGTTTCACAAGATATATTTGGTAGATTCATGGATGAAAAGGGCACATTAGAGAACCACCATTTCGCGCATTTAAAAGGAATGCTGGAACTTTTCGAGGCCTCAAACCTGGGTTTCGAAGGTGAAGATATTTTAGATGAGGCGAAAGCTTCCTTGACGCTAGCTCTCAGAGATAGTGGTCATATTTGTTATCCAGACAGTAACCTTTCCAGGGACGTAGTTCATTCCCTGGAGCTTCCATCACACCGCAGAGTGCAGTGGTTTGATGTCAAATGGCAAATCGACGCCTATGAAAAAGACATTTGTCGCGTCAACGCCACGTTACTCGAATTAGCAAAGCTTAATTTCAACGTAGTTCAGGCCCAACTCCAAAAAAACTTAAGGGAAGCATCCAGGTGGTGGGCAAACCTGGGCATCGCAGACAACTTGAAATTTGCAAGAGATAGACTGGTTGAATGTTTCGCATGTGCTGTGGGAGtAGCATTCGAGCCAGAGCACTCATC (SEQ ID NO:7)KLELIDSVRKLGLANLFEKEIKEALDSVAAIESDNLGTRDDLYATALHFKILRQHGYKVSQDIFGRFMDEKGTLENHHFAHLKGMLELFEASNLGFEGEDILDEAKASLTLALRDSGHICYPDSNLSRDVVHSLELPSHRRVQWFDVKWQIDAYEKDICRVNATLLELAKLNFNVVQAQLQKNLREASRWWANLGIADNLKFARDRLVECFACAVGVAFEPEHS

As a result of the identification of the polypeptides andpolynucleotides of the invention alpha-farnesene activity may bemodulated in plants. Modulation may involve a reduction in theexpression and/or activity (i.e. silencing) of the polypeptide.

Any conventional technique for effecting such silencing can be employed.Intervention can occur post-transcriptionally or pre-transcriptionally.Further, intervention can be focused upon the gene itself or onregulatory elements associated with the gene and which have an effect onexpression of the encoded polypeptide. “Regulatory elements” is usedhere in the widest possible sense and includes other genes whichinteract with the gene of interest.

Pre-transcription intervention can involve mutation of the gene itselfor of its regulatory elements. Such mutations can be point mutations,frameshift mutations, insertion mutations or deletion mutations. Socalled “knock-out” mutations in which expression of the gene can beentirely ablated. Alternatively transposon tagging may be used. Anotherapproach is to modify transcription through expression of a naturallyoccurring and/or artificial transcription factor, for example anartificial zinc finger protein transcription factor designed to interactwith the endogenous promoter of the alpha-farnesene synthase gene (seefor example http://www.sangamo.com/tech/tech.html.

Examples of post-transcription interventions include co-suppression oranti-sense strategies, a dominant negative approach, or techniques whichinvolve ribozymes to digest, or otherwise be lethal to, RNApost-transcription of the target gene.

Co-suppression can be effected in a manner similar to that discussed,for example, by Napoli et al. Plant Cell 2, 279-290 (1990) and deCarvalho Niebel et al. Plant Cell 7, 347-358 (1995). In some cases, itcan involve over-expression of the gene of interest through use of aconstitutive promoter. It can also involve transformation of a plantwith a non-coding region of the gene, such as an intron from the gene or5′ or 3′ untranslated region (UTR).

Anti-sense strategies involve expression or transcription of anexpression/transcription product capable of interfering with translationof mRNA transcribed from the target gene. This will normally be throughthe expression/transcription product hybridising to and forming a duplexwith the target mRNA.

The expression/transcription product can be a relatively small moleculeand still be capable of disrupting mRNA translation. However, the sameresult is achieved by expressing the whole polynucleotide in ananti-sense orientation such that the RNA produced by transcription ofthe anti-sense oriented gene is complementary to all or part of theendogenous target mRNA.

Anti-sense strategies are described generally by Robinson-Benion et al.Methods in Enzymol 254, 363-375 (1995) and Kawasaki et al., Artific.Organs 20, 836-845 (1996).

Genetic constructs designed for gene silencing may include an invertedrepeat. An ‘inverted repeat’ is a sequence that is repeated where thesecond half of the repeat is in the complementary strand, e.g.,

5′-GATCTA . . . TAGATC-3′ 3′-CTAGAT . . . ATCTAG-5′

The transcript formed may undergo complementary base pairing to form ahairpin structure provided there is a spacer of at least 3-5 bp betweenthe repeated regions.

Another approach is to develop a small antisense RNA targeted to thetranscript equivalent to an miRNA (Llave et al., Science 297, 2053-2056(2002) that could be used to target gene silencing.

The ribozyme approach to regulation of polypeptide expression involvesinserting appropriate sequences or subsequences (eg. DNA or RNA) inribozyme constructs (McIntyre Transgenic Res. 5 257-262 (1996)).Ribozymes are synthetic RNA molecules that comprise a hybridizing regioncomplementary to two regions, each of which comprises at least 5contiguous nucleotides of a mRNA molecule encoded by one of theinventive polynucleotides. Ribozymes possess highly specificendonuclease activity, which autocatalytically cleaves the mRNA.

Also contemplated is the use of dicer technology (Stratagene)

Alternately, modulation may involve an increase in the expression and oractivity of the polypeptide by over-expression of the correspondingpolynucleotide, or by increasing the number of copies of thecorresponding polynucleotide in the genome of the host.

As discussed in retention a gene silencing, approaches forover-expression may focus on the gene itself or on regulatory elementsassociated with the gene and which have an effect on expression of theencoded polypeptide. “Regulatory elements” is used here in the widestpossible sense and includes other genes which interact with the gene ofinterest. Another approach is to modify transcription through expressionof a naturally occurring and/or artificial transcription factor, forexample an artificial zinc finger protein transcription factor designedto interact with the endogenous promoter of the alpha-farnesene synthasegene (see for example http://www.sangamo.com/tech/tech.html.

The term “genetic construct” refers to a polynucleotide molecule,usually double-stranded DNA, which may have inserted into it anotherpolynucleotide molecule (the insert polynucleotide molecule) such as,but not limited to, a cDNA molecule. A genetic construct may contain thenecessary elements that permit transcribing the insert polynucleotidemolecule, and, optionally, translating the transcript into apolypeptide. The insert polynucleotide molecule may be derived from thehost cell, or may be derived from a different cell or organism and/ormay be a recombinant polynucleotide. Once inside the host cell thegenetic construct may become integrated in the host chromosomal DNA. Thegenetic construct may be lined to a vector.

To give effect to the above strategies, the invention also providesgenetic constructs usually DNA constructs. The DNA constructs includethe intended DNA (such as one or more copies of a polynucleotidesequence of the invention in a sense or anti-sense orientation or apolynucleotide encoding the appropriate ribozyme), preferably a promotersequence and preferably a termination sequence (which control expressionof the gene), operably linked to the DNA sequence to be transcribed. Thepromoter sequence is generally positioned at the 5′ end of the DNAsequence to be transcribed, and is employed to initiate transcription ofthe DNA sequence. Promoter sequences are generally found in the 5′non-coding region of a gene but they may exist in introns (Luehrsen Mol.Gen. Genet 225, 81-93 (1991)) or in the coding region.

A variety of promoter sequences which may be usefully employed in theDNA constructs of the present invention are well known in the art. Thepromoter sequence, and also the termination sequence, may be endogenousto the target plant host or may be exogenous, provided the promoter andterminator are functional in the target host. For example, the promoterand termination sequences may be from other plant species, plantviruses, bacterial plasmids and the like. Preferably, promoter andtermination sequences are those endogenously associated with thealpha-farnesene synthase genes.

Factors influencing the choice of promoter include the desired tissuespecificity of the construct, and the timing of transcription andtranslation. For example, constitutive promoters, such as the 35SCauliflower Mosaic Virus (CaMV 35S) promoter, will affect thetranscription in all parts of the plant. Use of a tissue specificpromoter will result in production of the desired sense or antisense RNAonly in the tissue of interest. With DNA constructs employing induciblepromoter sequences, the rate of RNA polymerase binding and initiationcan be modulated by external stimuli, such as chemicals, light, heat,anaerobic stress, alteration in nutrient conditions and the like.Temporally regulated promoters can be employed to effect modulation ofthe rate of RNA polymerase binding and initiation at a specific timeduring development of a transformed cell. Preferably, the originalpromoters from the gene in question, or promoters from a specifictissue-targeted gene in the organism to be transformed are used. Otherexamples of promoters which may be usefully employed in the presentinvention include, mannopine synthase (mas), octopine synthase (ocs) andthose reviewed by Chua et al. Science 244, 174-181 (1989).

The termination sequence, which is located 3′ to the DNA sequence to betranscribed, may come from the same gene as the promoter sequence or maybe from a different gene. Many termination sequences known in the artmay be usefully employed in the present invention, such as the 3′ end ofthe Agrobacterium tumefaciens nopaline synthase gene. However, preferredtermination sequences are those from the original gene or from thetarget species to be transformed.

The DNA constructs of the present invention may also contain a selectionmarker that is effective in cells, to allow for the detection oftransformed cells containing the construct. Such markers, which are wellknown in the art typically confer resistance to one or more toxins. Oneexample of such a marker is the NPTII gene whose expression results inresistance to kanamycin or hygromycin, antibiotics which are usuallytoxic to plant cells at a moderate concentration. Alternatively, thepresence of the desired construct in transformed cells can be determinedby means of other techniques well known in the art, such as PCR orSouthern blots.

Techniques for operatively linking the components of the inventive DNAconstructs are well known in the art and include the use of syntheticlinkers containing one or more restriction endonuclease sites. The DNAconstruct may be linked to a vector capable of replication in at leastone system, for example, E. coli, whereby after each manipulation theresulting construct can be sequenced and the correctness of themanipulation determined.

The DNA constructs of the present invention may be used to transform avariety of plants including agricultural, ornamental and horticulturalplants. In a preferred embodiment, the DNA constructs are employed totransform apple, banana, kiwifruit, tomato, cotton, rose, olive, potato,carnation, petunia, mango, papaya, lisianthus, chrysanthemum, rice, tea,hops and orchid plants.

As discussed above, transformation of a plant with a DNA constructincluding an open reading frame comprising a polynucleotide sequence ofthe invention wherein the open reading frame is orientated in a sensedirection can, in some cases, lead to a decrease in expression of thepolypeptide by co-suppression. Transformation of the plant with a DNAconstruct comprising an open reading frame or a non-coding(untranslated) region of a gene in an anti-sense orientation will leadto a decrease in the expression of the polypeptide in the transformedplant.

It will also be appreciated that transformation of other non-plant hostsis feasible, including well known prokaryotic and eukaryotic cells suchas bacteria (e.g. E. coli, Agrobacterium), fungi, insect, and animalcells is anticipated. This would enable production of recombinantpolypeptides of the invention or variants thereof. The use of cell freesystems (e.g. Roche Rapid Translation System) for production ofrecombinant proteins is also anticipated (Zubay Annu Rev Genet 7,267-287 (1973)).

The polypeptides of the invention produced in any such hosts may beisolated and purified from same using well known techniques. Thepolypeptides may be used in cell-free systems for enzymic synthesis ofalpha-farnesene.

Techniques for stably incorporating DNA constructs into the genome oftarget plants are well known in the art and include Agrobacteriumtumefaciens mediated introduction, electroporation, protoplast fusion,injection into reproductive organs, injection into immature embryos,high velocity projectile introduction, floral dipping and the like. Thechoice of technique will depend upon the target plant to be transformed.

Once the cells are transformed, cells having the DNA constructincorporated into their genome may be selected by means of a marker,such as the kanamycin resistance marker discussed above. Transgeniccells may then be cultured in an appropriate medium to regenerate wholeplants, using techniques well known in the art. In the case ofprotoplasts, the cell wall is allowed to reform under appropriateosmotic conditions. In the case of seeds or embryos, an appropriategermination or callus initiation medium is employed. For explants, anappropriate regeneration medium is used.

In addition to methods described above, several methods are well knownin the art for transferring DNA constructs into a wide variety of plantspecies, including gymnosperms angiosperms, monocots and dicots.

The resulting transformed plants may be reproduced sexually orasexually, using methods well known in the art, to give successivegenerations of transgenic plants.

The nucleotide sequence information provided herein will also be usefulin programs for identifying nucleic acid variants from, for example,other organisms or tissues, particularly plants, and for pre-selectingplants with mutations in alpha-farnesene synthase or their equivalentswhich renders those plants useful. This provides for an acceleratedbreeding program to produce plants in which the content ofalpha-farnesene and its derivatives is modulated. More particularly, thenucleotide sequence information provided herein may be used to designprobes and primers for probing or amplification of alpha-farnesenesynthase. An oligonucleotide for use in probing or PCR may be about 30or fewer nucleotides in length. Generally, specific primers are upwardsof 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16-24 nucleotides in length are preferred.Those skilled in the art are well versed in the design of primers foruse in processes such as PCR.

If required, probing can be done with entire restriction fragments ofthe gene disclosed herein. Naturally, sequences based upon FIG. 4 or thecomplements thereof can be used. Such probes and primers also formaspects of the present invention.

Methods to find variants of the of polynucleotides of the invention fromany species, using the sequence information provided by the invention,include but are not limited to, screening of cDNA libraries, RT-PCR,screening of genomic libraries and computer aided searching of EST, cDNAand genomic databases. Such methods are well known to those skilled inthe art.

The invention will now be illustrated with reference to the followingnon-limiting Examples.

EXAMPLES

The following Examples further illustrate practice of the invention.

Example I Identification of the Alpha-farnesene Synthase Gene

Plant material and GC-MS analysis: Tree-ripened 150 DAFB apples (Malusdomestica) were harvested from Royal Gala trees grown in a HortResearchorchard at Hawkes Bay, New Zealand. Twelve fruit were selected foranalysis and were placed into a 5 L wide-necked round-bottomed samplingvessel with a ground glass flat flange joint. The vessel was coveredwith a glass lid with a sealed ground glass joint inlet socketcontaining a gas line and a volatile sorbent cartridge containing 100 mgChromosorb 105. The headspace in the flask was allowed to equilibrate at23° C. for 1 hour, after which the headspace was purged with N₂(g) at25.0 ml/min while being trapped for 15 min. The Chromosorb cartridge wasdried with a N₂(g) flow at 10 psi, 35 C for 15 min prior to analysis.The volatiles were thermally desorbed from the Chromosorb traps for 3min at 150° C. into the injection port of the gas chromatograph (GC)HP5890. The GC system was equipped with a DB-Wax capillary column (J & WScientific, Folsom, USA), 30 m×0.32 mm i.d., with a 0.5 μM filmthickness. The carrier gas was helium at a flow rate of 30 cm/sec. TheGC oven was programmed to remain at 30° C. for 6 min, then to increaseby 3° C./min to 102° C., followed by an increase of 5° C./min to 190°C., which was maintained for 5 min. The column outlet was split to amass spectrometer (VG70SE), in addition to the GC's flame ionisationdetector (GC-FID/MS). The mass spectrometer operated in electron impactionisation (EI-MS) mode at 70 eV with a scan range 30-320 amu. Componentidentification was assisted with mass spectra of authentic standards,library spectra (NIST and in-house) and GC retention indices.Quantitative data was obtained by measuring the sample peak areasrelative to an authentic standard.

Isolation of mRNA and cDNA library construction: The skin of the 150DAFB apples was removed with a peeler and total RNA was extracted fromthe peeled skin by an adaptation of the method of Gomez and Gomez(Langenkamper, et al., Plant Mol. Biol. 36, 857-869 (1998)). mRNApurified from the total RNA by oligo(dT)-cellulose chromatography(Pharmacia) was used to construct a Lambda ZAP-CMV (Stratagene) cDNAlibrary according to the manufacturer's instructions. ThecDNA-containing pBK-CMV plasmids were massed excised and used totransform E. coli XLOLR (Stratagene). The plasmids were isolated fromthe XLOLR colonies and partially sequenced. All sequences on thedatabase were BLASTed against the NRBD90 database (Altschul, et al.,Nucleic Acids Res. 25, 3389-3402 (1997).) and putative terpene synthasecDNA sequences were identified by their similarity to known terpenesynthases based on key motifs. A full-length terpene synthase sequence(EST57400) was identified and its polynucleotide sequence determined.

Cloning into pET-30: For functional expression, a cDNA fragment encodingEST57400 was excised from pBK-CMV57400 using a EcoRI restrictionendonuclease site immediately adjacent to the start ATG and the vectorXhoI restriction site. The resultant 1899 bp cDNA sequence was thensubcloned in frame into the expression vector pET-30a (Novagen), whichwas also digested with EcoRI and XhoI, yielding plasmid pET-30a57400.Plasmid pET-30a57400 was then transformed into E. coliBL21-CodonPlus™-RIL cells (Stratagene). The clone was resequenced at the5′ end to ensure the inserted cDNA was in frame.

Expression and characterization of alpha-farnesene synthase frombacterial cultures: E. coli BL21-Plus™-RIL cells harbouringpET-30a57400, and empty pET-30 vector as a control, were grown overnightat 37° C. in Lauria-Bertani media supplemented with 30 μg/ml kanamycinand 50 μg/ml chloramphenicol. A 500 μl aliquot of overnight culture wasused to inoculate 50 ml of fresh 2×YT medium supplemented with 30 μg/mlkanamycin and 50 μg/ml chloramphenicol. The culture was grown at 37° C.with vigorous agitation to A₆₀₀=0.6 before induction with 0.3 mMisopropyl-β-D-thiogalactopyranoside (IPTG) and simultaneous addition offarnesyl diphosphate (FDP) (10 μM). The culture was immediatelytransferred to a 30° C. incubator, or 16 or 37° C. incubators dependingon the experiment.

Headspace analysis of bacterial cultures: The headspace in the vesselsabove the bacterial cultures was collected immediately after theaddition of FDP using solid phase micro extraction (SPME). The SPMEfibres (65 μm PDMS/DVB, Supelco, Australia) were conditioned for 45 minat 260° C. and the background analysed for contamination using GC-FID(HP5890) prior to use. The headspace volatiles were collected for 4hours at 30° C. with continuous agitation (110 rpm). Prior to analysisusing a GC-FID/MS, the fibres were stored at ambient temperature inseptum sealed glass vials. The volatiles were desorbed from the fibresfor 5 minutes at 250° C. in the GC injection port. The GC system wasequipped with a DB-Wax capillary column (J & W Scientific, Folsom, USA),30 m×0.25 mm i.d., with a 0.5 μm film thickness. The carrier gas washelium at a flow rate of 30 cm/sec. The GC oven was programmed to remainat 30° C. for 6 min, then to increase by 3° C./min to 102° C., followedby an increase of 5° C./min to 210° C., which was maintained for 11 min.The mass spectrometer operated in electron impact ionisation (EI-MS)mode at 70 eV with a scan range of 30-320 amu. Peak identification wascarried out by comparison of sample spectra with those from NIST, Wiley,and our own mass spectra libraries and confirmed by retention indices ofauthentic standards and literature values (Davies, J. Chrom. 503, 1-24,(1990)). Quantitative data was obtained by measuring sample peak arearelative to an internal standard, hexadecane, which had been added tothe cultures at the same time as the FDP.

Expression time course for induced and non-induced cultures: 6×50 mlbacterial cultures harbouring pET-30a57400 were prepared as above. AtA₆₀₀=0.6 three of the cultures were induced with 0.3 mM IPTG leaving theremaining cultures non-induced. Cultures were then incubated for one,three or five hours at 30° C. and the headspace volatiles were collectedas described above.

Characterization of alpha-farnesene synthase from bacterial extracts andpartially purified alpha-farnesene synthase recombinant protein:Cultures were set up, grown and induced as above. Following induction,cultures were immediately transferred to a 24° C. incubator and allowedto grow for a further 18-20 hours with continuous agitation and thencells harvested by centrifugation (2000×g for 10 min). Pelleted cellswere resuspended in either 20 ml binding buffer (5 mM imidazole, 0.5 mMNaCl, 10 mM DTT, 20 mM Tris-HCl (pH 7.9) or 20 ml extraction buffer (25mM MOPS (pH 7.0), 10 mM sodium ascorbate, 25 mM KCl, 10 mM DTT, 10%glycerol). Cells were disrupted with 2× exposure to 12,700 psi in aFrench Pressure Cell Press (American Instrument Co. Inc, Silver Spring,Md. USA) and then centrifuged at 8000×g for 15 min. 5 ml of supernatantwas transferred to a 50 ml test-tube and adjusted to 10 mM MgCl₂ and 20μM MnCl₂. FDP (100 μM) was added and the reaction mixture was incubatedat 30° C. Headspace volatiles were collected as in the same manner aswhole cultures. The remainder of the extract (15 ml) was applied toPD-10 gel filtration columns (Amersham-Pharmacia Biotech)pre-equilibrated with either binding or extraction buffer (DTT omitted).Eluent fractions were then pooled and purification of recombinantprotein was carried out in a single step using immobilised metalaffinity chromatography (IMAC). The eluent was applied to a Hi-TrapChelating HP column (Amersham-Pharmacia Biotech) charged with Ni⁺. Nonbound proteins were removed and recombinant protein was eluted followingthe manufacturer's specifications. Five ml samples of the eluted proteinwere transferred to 50 ml test-tubes and adjusted to 10 mM MgCl₂, 20 μMMnCl₂ and 10 μM FDP was added. Headspace volatiles were collected as inthe bacterial cultures. Aliquots of the remaining recombinant proteinwere stored at −80° C. in 20% glycerol until required.

Electrophoresis and Western analysis: Whole culture, French PressHis-purified and non His-purified protein extracts were analysed bySDS-PAGE, using 10% polyacrylamide gels. Protein bands were eithervisualised using Colloidal Coomassie or were transferred on toImmobilin-P PVDF membrane (Millipore). Blotted proteins were incubatedwith Anti-His₆ monoclonal (Roche) primary and Anti-Mouse IgG-AP(Stressgen) secondary antibodies and were detected using 1-STEP™NBT/BCIP (Pierce) alkaline phosphatase detection reagent.

Protein quantification: Protein concentrations of extracts and partiallypurified recombinant proteins were determined according to Bradfordusing the Biorad kit according to manufacturers specification using aSpectromax Plus spectrophotometer, using bovine serum albumin (BSA) asthe standard.

Results

Headspace analysis of volatiles emitted from 150 DAFB apples: It is wellestablished that alpha-farnesene is synthesised in apple skin tissue anddetected in headspace analyses. Typically two isomers of alpha-farneseneare found in apple skin, (E,E) and (Z,E) alpha-farnesene (Matich, etal., Anal. Chem. 68, 4114-4118 (1996), Bengtsson, et al., J. Agric. FoodChem. 49, 3736-3741 (2001)). These two isomers are usually identified inthe ratio of 100:1 respectively (Matich, et al., Anal. Chem. 68,4114-4118 (1996)). In the headspace of the 150 DAFB apples analysed onlythe ‘all trans’ (E,E) isomer of alpha-farnesene was identified (FIG. 5).This isomer was present at low levels, on average 4 ng (E,E)alpha-farnesene per fruit. The (E,E) alpha-farnesene isomer had aretention time of 42.57 minutes that was used to calculate the Kovatsretention index for this compound. The retention index and the massspectra positively identified this compound as (E,E) alpha-farnesene.

Sequence analysis of alpha-farnesene synthase: Sequencing of the cDNA inpBK-CMV that encoded alpha-farnesene synthase revealed an insert size of1926 base pairs excluding the poly(A) tail (FIG. 3, SEQ ID NO:1). ThecDNA sequence had a predicted ORF of 576 amino acids beginning with aputative start methionine 61 bases in from the 5′ end (FIG. 4, SEQ IDNO:2). The molecular mass of alpha-farnesene synthase is predicted to be66 kD. The predicted amino acid sequence of alpha-farnesene synthasedoes not have a chloroplast-signalling peptide sequence (Emanuelsson, etal., 300, 1005-1016 (2000)), which is typical of monoterpene andditerpene synthases. As has been found for all other terpene synthasesthe predicted amino acid sequence of alpha-farnesene synthase contains aDDXX(D,E) motif (DDVYD) at amino acids 326 to 330 that is involved inthe binding of the metal ions necessary for catalysis. alpha-farnesenesynthase was not shown to contain the angiosperm sesquiterpene consensussequence GVYXEP (Cai et al Phytochem 61, 523-529 (2002)), insteadcontaining a highly similar GVAFEP motif from amino acids 301 to 306. Itwas also shown to contain the RRX₈W motif at amino acids 33 to 43, whichis a common characteristic of Tps-d and Tps-b monoterpene synthases.(Duderava, N., Martin, D., Kish, C. M., Kolosova, N., Gorenstein, N.,Fäldt, J., Miller, B., and Bohlmann, J. (2003) Plant Cell. 15,1227-1241.)

Bohlmann, Meyer-Gauen and Croteau (Proc. Natl. Acad. Sci. USA 95,4126-4133 (1998)) compared the amino acid sequences of 33 terpenesynthases and showed that there were seven absolutely conserved aminoacid residues. Alpha-farnesene synthase contains six of these sevenabsolutely conserved amino acids. They also found that six positionswere absolutely conserved for aromatic amino acids and four positionswere absolutely conserved for acidic amino acids. In alpha-farnesenesynthase, four of the six aromatic positions and all of the four acidicpositions are conserved.

The predicted amino acid sequence for alpha-farnesene synthase (asesquiterpene synthase) most closely resembles the amino acid sequencesof a putative monoterpene from Cinnamomum tenuipilum (Zeng et al,Genbank CAD29734, 2002), having 39.8% identity and 56.3% similarity fromthe predicted amino acids 34 to 574. A putative monoterpene synthasefrom Melaleuca alternifolia (tea tree) (Shelton et al, GenebankAAP40638, 2003) has the second highest similarity, with 38.7% identityand 54.1% similarity from the predicted amino acids 34 to 574 and aputative monoterpene synthase from Quercus ilex (holly oak) (Fischbach,Genbank CAC41012, 2001), having 37.9% identity and 55.8% similarity fromthe predicted amino acids 34 to 574.

The nucleic acid sequences of the alpha-farnesene synthase show homologyto very short stretches of the mRNA of a few sesquiterpene synthases.One area of homology lies between nucleotides 918 and 946. For example,cadinene synthase from Gossypium arboreum (tree cotton) (Chen, Wang,Chen, Davisson and Heinstein, 1996) has 24 out 25 identical bases in theregion between nucleotides 918 and 946 and a putative sesquiterpenesynthase from Artemisia annua has 25 out 26 bases identical in thisregion (Van Geldre et al., Plant Sci. 158, 163-171 (2000)). Betweennucleotides 367 and 386, E- a-bisabolene synthase of Abies grandis(grand fir) has 20 out of 20 identical bases.

The predicted isoelectric point for alpha-farnesene synthase synthase is5.1 which is similar to the isoelectric point calculated for othersesquiterpene synthases. For example, two sesquiterpene synthasesisolated from Artemisia annua, cASC34 and cASC125, have isoelectricpoints of 5.28 and 5.50, respectively (Van Geldre et al., Plant Sci.158, 163-171 (2000)).

The cDNA sequence for alpha-farnesene synthase (EST 57400) was obtainedfrom a cDNA library constructed from Royal Gala 150 DAFB apple skin.Three other truncated cDNAs with polynucleotides across the sequenced 5′end identical to EST 57400 were also isolated. One was from Royal Gala126 DAFB fruit cortex, one from Royal Gala floral buds, and the thirdtruncated one from Pinkie leaf. Another truncated cDNA obtained fromAotea leaf had seven base pair differences out the 675 bases sequenced,resulting in 5 amino acid changes

Western analysis: Western analysis confirmed the presence of a solubleexpression product within the expected size range (65-75 kDa His taginclusive) for alpha-farnesene synthase in both the French Pressextracts and partially purified recombinant protein extracts. Nosimilar-sized band was detected in either the pET-30a control purifiedor non-purified extracts.

Characterisation of alpha-farnesene synthase (E,E)-alpha-farnesene andsmall amounts (Z,E)-alpha-farnesene were detected in the headspace ofbacterial cultures and extracts harbouring pET-30a57400. Controlscomprising E. coli BL21 cells transformed with pET-30a lacking thealpha-farnesene synthase cDNA insert gave negligible or noalpha-farnesene. (E,E)-alpha-farnesene production in both cultures andcrude extracts, although not dependent on precursor addition, was shownto be dependent on the presence of the alpha-farnesene synthase cDNAinsert. Although a peak was found at a similar retention time in thecontrol as the alpha-farnesene (42.24 min), the mass spectra showed thisto be citral. Addition of GDP to bacterial cultures did not produceeither alpha-farnesene or any monoterpenes.

Headspace analysis of partially purified recombinant enzyme, whetherderived from extraction in His purification binding buffer orsesquiterpene extraction buffer, showed (E,E)-alpha-farnesene as themajor product with minor amounts of (Z,E)-alpha-farnesene present (FIG.6). This required added FDP, no alpha-farnesene was produced without theadded precursor. Purified enzyme that had been stored in glycerol for 4weeks at −80 C was reassayed with only 15% loss of activity.

The bacterial cultures and crude extracts harbouring the alpha-farnesenecDNA showed leaky expression under non-inducing conditions. Howevervolatile trapping over a 5 hour period demonstrated that addition ofIPTG increased the production of both isomers relative to the samplesthat were not induced.

EST 57400 therefore encodes an alpha-farnesene synthase that makes onlyalpha-farnesene.

Example II Properties of the Enzyme

Isomer Specificity: FDP precursor consisted of a mixture of E,E and E,Zforms in most experiments and analyses indicated that both E,E and Z,Eisomers of alpha-farnesene were produced by the gene alpha-farnesenesynthase. To test isomer specificity we fed FDP precursor in the ratios41.3% E,E isomer, 28.7% E,Z isomer, 24.7% Z,E and 5.4% Z,Z to purifiedprotein extracts in a standard in vitro activity experiment. Following 2h trapping with SPME fibres as earlier described three of the fourisomers of alpha-farnesene were produced (see FIG. 7). The peak at 40.54may be Z,Z alpha-farnesene but it has not been confirmed. These resultsindicate the enzyme has no isomeric specificity and will producealpha-farnesene isomers dependent on the isomeric form of the FDPprecursor.

Optimisation of large-scale production of protein: E. coli BL21-Plus™-RIL cells harbouring pET-30a57400 were grown overnight at 37° C. inLauria-Bertani media supplemented with 30 μg ml⁻¹ kanamycin and 50 μg mlchloramphenicol⁻¹. 5 mL aliquots of overnight culture were used toinoculate 4×300 mL of fresh 2×YT medium supplemented with 30 μg ml⁻¹kanamycin and 50 μg ml⁻¹ chloramphenicol in 1 L baffled flasks. Cultureswere grown at 37° C. with vigorous agitation to A₆₀₀=0.8, then removedto 4° C. to equilibrate to 16° C. before induction with 0.3 mM IPTG.Induced cultures were then incubated at 16° C. and 220 rpm for a further50 hours. Cells were pelleted by centrifugation (2500×g; 10 min) andstored overnight at −20° C. The following day cell pellets wereresuspended in 15 mL His6 binding buffer and cells were disrupted by 3passes through an EmulsiFlex®-C15 high-pressure homogeniser (Avestin)with a pressure setting between 15000-20000 psi. Cell debris waspelleted by centrifugation 2× at 10000×g for 15 min; 4° C. (Sorval SS34rotor). The supernatant was filtered through a 0.45 μm filter (Amicon).Filtered extract was desalted and passed over a Nickel affinity column,followed by passage through a 30 kDa filter (Millipore).

Protein concentration was determined from the extinction coefficient(VectorNTI version 8) and the purified extract adjusted to ˜1 mgprotein/mL with His6 elution buffer containing 10% glycerol and 1 mMDTT. The extract was then separated into 100 μl aliquots and stored at−80° C. until required.

Protein oligomerisation: Approximately 500 μg of purified protein wasloaded onto a 600×16 mm S300 Sephacryl column (Pharmacia) at a flow rateof 1 mL min⁻¹. The column was preequilibrated and eluted with 50 mMBis-tris propane buffer containing 10% glycerol with either 50 mM KCl or0.5 M KCl with or without 7 mM MgCl₂. Fractions corresponding to proteinpeaks were assayed after adjusting to 7 mM MgCl₂ for activity afteraddition of 25 μM ³H-FDP. Molecular mass of active fractions wascalculated based on comparison to standards of known molecular mass.

Results: The alpha-farnesene synthase protein acts primarily as amonomer (see FIGS. 8 and 9). While the data suggests a small amount ofactivity could be due to oligomeric forms, over 70% of the activity isdue to monomeric enzyme. FIG. 8 shows the protein elution profile andapproximate molecular mass of alpha-farnesene synthase on SephacrylS-300 HR Gel filtration chromatography. Four different purified extractswere compared with contrasting salt and Mg profiles. The major peak atfraction 40 is not alpha-farnesene synthase, shows almost no protein onan SDS gel and is likely to be predominantly DNA; enzymatic activitycenters on fractions between 60 and 70 (see FIG. 9).

Kinetic Studies: For kinetic studies, alpha-farnesene synthase activeprotein that had been induced in culture and purified as described forprotein optimisation was added to a minimal assay buffer containing 50mM Bis-Tris-Propane (pH 7.5), 10% (v/v) glycerol, 1 mM DTT and 0.1%(v/v) Tween-20. Radioactive FDP was added variously depending on theexperiment. One mL assays containing 1-2 μg of protein were overlaidwith 0.6 mL pentane and incubated in 1.5 mL microfuge tubes for 2 hoursat 30° C. and 150 rpm. All assays were performed in triplicate.Following incubation assays were immediately placed on ice and a 200 μLaliquot of the pentane layer removed for analysis. The aliquots wereadded to 1.5 mL microfuge tubes containing 0.7 DL Organic CountingScintillant (OCS) (Amersham) and vortexed briefly. Scintillationanalysis was performed using a Wallac 1409 Liquid Scintillation Counter(³H efficiency≈70%).

Kinetic studies with ³H-FDP (10.06 Mbq/mL) as substrate (concentrationrange 1 μm to 100 μM with saturating Mg²⁺ and Mn²⁺) were carried out todetermine Km for FDP. Kinetic constants for Mg²⁺, and Mn²⁺ at 25 μM³H-FDP (assay range 25 μM to 25 mM and 1 μM to 1 mM of the chloridesalts respectively) were determined. The effect on enzyme activity ofmetal co-factors with and without salts was also tested. Mg²⁺ and Mn²⁺were added in the presence and absence of 50 mM KCL and 50 mM NaCl inall possible combinations. Controls included incubation without enzyme,with enzyme but without metal ion cofactors and with enzyme andcofactors in the presence of 10 mM EDTA.

For determination of the enzyme pH optimum, assays were carried in atri-buffer system containing 51 mM diethanolamine, 100 mM MES, and 51 mMN-ethylmorpholine at pH values between 4.5 and 9.6 with 7 mM Mg, 150 μMMn and 25 μM FDP, 10% (v/v) glycerol, 1 mM DTT and 0.1% (v/v) Tween-20.Optimal temperatures for enzyme activity in the range 18° C.-50° C. werealso determined using the standard assay buffer and 7 mM Mg, 150 μM Mnand 25 μM FDP. Kinetic constants were determined from the DPM data bynonlinear regression using the Origin50 graphics package. Data presentedrepresents the means of three determinations with standard errors within±10% and with background DPM calculated from controls subtracted. Allexperiments were carried out at least twice.

Results: pH alpha-farnesene synthase showed a broad pH optimum betweenpH7 and 8.5 (FIG. 10). Repeated experiments indicated a slight reductionin activity at ˜pH7.8. There was no difference in product produced overthe optimal pH range for activity (data not shown). This pH range issimilar to that (between pH 7 and 9)reported for other characterisedsesquiterpene synthases in the literature (Cai et al Phytochemistry 61,523-529 (2002); Steele et al J. Biol. Chem. 273, 2078-2089 (1998))

Km The Km for FDP was between 2.5 and 3.5 μM (FIG. 11) with saturatingconcentrations at 12 μM. No precursor inhibition was found atconcentrations of FDP up to 50 μM. Km for the cofactor Mg²⁺ was between600 and 800 μM (FIG. 12) with a slight inhibition (23%) of activity withhigh (25 mM) concentrations; and the Km for Mn²⁺ was between 10 and 20μM with at least 50% inhibition of activity at high (>800 μM)concentrations (FIG. 13). Reported Kms in the literature range from0.4-4.5 μM for FDP, 70-150 μM for Mg, 7-30 μM for Mn for othersesquiterpene synthases (eg Cai et al Phytochemistry 61, 523-529 (2002),Steele et al J. Biol. Chem. 273, 2078-2089(1998)).

Other metal ion effects Activity of alpha-farnesene synthase wasenhanced with addition of K⁺ ions (Table 1). Addition of Na⁺ ionsresulted in a slight non significant enhancement of activity, indicatingthe enhancement was due to the form of metal ion and not a general saltresponse.

TABLE 1 Relative activity of alpha-farnesene synthase due to additionsof metal ions in the presence of saturating FDP. Metal ion V_(rel) (%)Mg/KCl (7 mM/50 mM) 100  Mn/KCl (150 μM/50 mM) 41 Mg/Mn/KCl (7 mM/150μM/ 69 50 mM) Mg (7 mM) 16 Mn (150 μM) 13 Mg/Mn (7 mM/150 μM) 18 Mg/NaCl(7 mM/50 mM) 23 Mn/NaCl (150 μM/50 mM) 15 Mg/Mn/NaCl (7 mM/150 24 μM 50mM)

Temperature Maximum alpha-farnesene synthase activity occurred at 37°C., with a sharp reduction in activity at higher temperatures (FIG. 14).Activity was not detectable at 50° C., whereas at low temperatures (13°C.) enzyme activity remained although reduced by two thirds. This issimilar to that reported for other sesquiterpene synthases.

Storage

Protein stored at −80° C. for 9 months lost activity (92.5% loss). Thisstored protein also showed a decrease in Km for FDP to ˜1.5 μM. HoweverKm for Mg, Mn and the pH response were unchanged.

Example III Expression in Plants

Transient expression in Nicotiana benthamiana leaves: Transformation ofN. benthamiana leaves was performed according to Hawes et al. (Hawes,C., Boevink, P., and Moore, I., GFP in plants, in Fluorescencemicroscopy of proteins: a practical approach., V. Allen, Editor. 1999,Oxford University Press: Oxford. p. 163-177 (2000)). Agrobacteriumtumefaciens strain GV3101 (MP90) containing pHEX2 binary vectorharbouring EST 57400, and P19 vector expressing a viral suppressor ofmRNA silencing (Voinnet et al. Plant J 33, 949-956 (2003)) as a controlwere grown in 5 ml cultures of 2YT media containing rifampicin (10 mgml⁻¹); gentamycin (25 mg ml⁻¹); and spectinomycin (100 mg ml⁻¹) for 24hours at 28° C. The cells were collected by centrifugation (3,500×g; 10minutes), and resuspended in infiltration medium (50 mM MES pH5.6, 0.5%(w/v) glucose, 2 mM Na₃PO₄, 100 mM acetosyringone (made freshly from 200mM acetosyringone/DMSO stock)) to a final OD₆₀₀ of 0.5-0.6. Thebacterial suspension was injected through the stomata on the undersideof detached N. benthamiana leaves, using a 1 ml syringe with no needleattached. The infiltrated area on the leaf was marked with an indeliblemarker pen for later identification. Plants with infected leaves weregrown for 7 days under standard green house conditions.

Transformed N. benthamiana leaves and control leaves were infiltratedwith distilled water containing 25 μM FDP, 7 mM MgCl₂ and 50 mM KCl. Onehour later, 3 leaves per treatment were excised and placed in 50 mLglass test tubes and the headspace above the leaf tissue was collectedusing solid phase micro extraction (SPME) for 12 hours at 30° C. Theexperiment was repeated without preinfiltration. The complete experimentwas carried out on two occasions with independently transformed plants.

Results:

Transient expression studies show that alpha-farnesene is produced whenthe alpha-farnesene synthase gene is present but not produced in thecontrol leaves. E,E alpha-farnesene was produced without the addition ofthe precursor (suggesting that some endogenous precursor is available inthe N. benthamiana leaves) however, more E,E alpha-farnesene wasproduced when the precursor was also infiltrated into the leaves. Theresults are shown in FIG. 15.

Over-expression of apple alpha-farnesene synthase in Arabidopsis: A.tumefaciens, strain GV3101, was inoculated into 10 mL of LB mediacontaining rifampicin (10 mg mL⁻¹); gentamycin (25 mg mL⁻¹); andspectinomycin (100 mg mL⁻¹) and grown for 24 hours at 28° C., withshaking at 200 rpm. This starter culture was then used to inoculate afurther 100-200 mL of LB media containing antibiotics as above. This wasagain grown for 24 hours at 28° C., with shaking. The cells werecollected by centrifugation (3,500×g, 10 min, 4° C.) and resuspended in5% sucrose solution, to a final OD₆₀₀ of 0.8. Silwet L-77 was added to aconcentration of 0.05%. 45 mL aliquots of these competent Agrobacteriumcells were thawed gently on ice. 50-200 ng of plasmid DNA (pHEX2 vectorharbouring EST57400) was added to each aliquot and gently mixed, then 40mL of the cell/plasmid mixture was pipetted into a pre-chilledelectroporation cuvette (0.2 cm gap, Bio-Rad). The cells wereelectroporated using a BioRad GenePulser, on the following settings:

-   Voltage: 2.5 kV-   Capacitance: 25 mFd-   Resistance: 400 Ohms

The time constant for the pulse was typically 7-9 ms.

The cells were immediately recovered by addition of 1 mL LB media, thendecanted into sterile 15 mL centrifuge tubes and incubated at roomtemperature, with shaking (60 rpm). After 2 hours, 10 mL and 100 mL ofthe transformed bacteria was spread onto separate LB plates containingrifampicin (10 mg mL⁻¹); gentamycin (25 mg mL⁻¹); and spectinomycin (100mg mL⁻¹); then grown for 48 hours at 28-30° C.

A. tumefaciens, containing the plasmid, was grown in 5 mL cultures of LBmedia containing rifampicin (10 mg mL⁻¹); gentamycin (25 mg mL⁻¹); andspectinomycin (100 mg mL⁻¹) for 24 hours at 28° C. The cells werecollected by centrifugation and resuspended in 5% sucrose solution, to afinal OD₆₀₀ of 0.8. Silwet L-77 was added to a concentration of 0.05%.

Healthy five week old Arabidopsis thaliana cv Columbia plants, showing anumber of immature flower clusters, were transformed with the EST57400containing or empty vector-containing Agrobacterium. The whole of theaboveground portion of the plant was dipped into the Agrobacteriumsuspension, and gently agitated for 3-5 seconds. The dipped plants werethen placed in humidity chambers in reduced light for 2-3 days, beforebeing allowed to flower and set seed under normal glasshouse conditions.The seed (T0) was harvested upon complete drying of the plants (5-6weeks after dipping). T2 seed was generated by selfing plants andgrowing individuals on kanamycin selection plates over two generations.

DNA extraction: For southern analysis, DNA was extracted fromArabidopsis thaliana leaves by the method of Dellaporta et al. PlantMol. Biol. Reporter 1, 19-21 (1983). Tissue material (1 g) was ground inliquid nitrogen and then the powder was added to 15 ml buffer (100 mMTris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl) containing 22.5 μlβ-mercaptoethanol. After the addition of 1 ml of 20% SDS, the homogenatewas touch vortexed and then incubated at 65° C. for 20 minutes. Cold 5 Mpotassium acetate (5 ml) was added, followed by incubation on ice for 20minutes and then centrifugation at 6000 rpm for 30 minutes. Thesupernatant was passed through miracloth, 10 ml cold isopropanol wasadded and the DNA was left to precipitate overnight at 4° C. The DNA waspelleted by centrifugation at 5000 rpm for 15 minutes, then washed in 1ml 70% ethanol and resuspended in 0.5 ml water. The resuspended DNA wasextracted with an equal volume of 1:1 phenol:chloroform, then extractedwith an equal volume of chloroform and reprecipitated with isopropanol.After centrifugation and washing in 70% ethanol the DNA was resuspendedin 50 μl water. RNAse (Roche) (1 μl of 10 mg/ml) was added to removeRNA.

For small-scale DNA extractions used in PCR reactions, 150 mg ofArabidopsis thaliana leaf tissue was ground in liquid nitrogen.Extraction buffer (480 μl) was added and the tissue was ground furtherand then left on ice. After adding 37.5 μl of 20% SDS the samples wereput at 65° C. for 10 minutes. The samples were mixed by inversion afteradding 94 μl of 5 M potassium acetate and then left in ice for 10minutes before centrifugation at 13,200 rpm for 10 minutes. Thesupernatant was extracted with 600 μl 25:24:1 phenol:chloroform:isoamylacetate with gentle mixing and then centrifuged at 10,000 rpm for 5minutes. 360 μl of isopropanol was added to the supernatant, mixed andthen centrifuged for 3 minutes at 13,200 rpm. The pellet was rinsed with70% ethanol, centrifuged for 1 minute, rinsed with 95% ethanol,air-dried and resuspended in 50 μl TE containing RNAse (Roche).

PCR amplification of DNA PCR amplifications were undertaken using ExpandHigh Fidelity Taq (Roche). Amplification reactions were performedaccording to the manufacturer's recommendations. The PCR primers(57400_A3 AGAGTTCACTTGCAAGCTGA (SEQ ID NO:3) and 57400_A4GAAAAGTTCCAGCATTCCTT) (SEQ ID NO:8) were designed to amplify a 513 basepair fragment from the 5′ end of the coding region of EST 57400. PCRamplification was performed under the following conditions: denaturationat 96° C. for 5 minutes followed by 30 cycles of denaturation at 96° C.for 30 seconds, annealing at 55° C. for 4 seconds and extension at 72°C. for 60 seconds. The resulting amplification products (5 μl) wereanalysed by electrophoresis through 1% AppliChem, followed by stainingwith ethidium bromide and visualisation on an ultraviolettransilluminator (UVP) attached to a camera (UV tec, Total Lab SystemsLtd).

Results: DNA encoding alpha-farnesene synthase from apple was detectedby PCR in each of the four transgenic Arabidopsis independenttransformants (FIG. 16) and in apple controls. None was detected in thepHEX control.

Southern Analysis

Genomic DNA that had been extracted from Arabidopsis thaliana leaftissue was digested overnight with BamH1 in a total volume of 500 μl.The digested DNA was precipitated with two volumes of ethanol andone-tenth volume of 3M sodium acetate, then centrifuged, washed in 70%ethanol and the pellet resuspended in 30 μl water. The digested DNA wasthen electrophoresed through 0.7% agarose, visualised with ethidiumbromide, hydrolysed in 0.25M HCl and washed in water before transfer toNytran-Plus (Schleicher & Schuell) membrane in 0.4M NaOH overnight. Themembrane was then neutralised in 0.5M Tris and prehybridised in Washingand Pre-Hyb Solution (MRC) for two hours. Hybridisation was performed in20 ml of High Efficiency Hybridzation System (MRC) using as a probe a³²P-labelled 810 base pair PCR fragment that was complementary to the 5′end of the coding region of alpha-farnesene synthase. The probe (40 ng)was labelled with ³²P dCTP using the rediprime™II (Amersham Pharmacia)random labelling system, following the manufacturer's directions. Thelabelled probe was denatured in 0.1 M NaOH for 30 minutes beforehybridisation overnight. The membrane was washed in Washing and Pre-HybSolution (MRC) according to the manufacturer's recommendations.Hybridisation signals were visualised by scanning on a Storm 840phospho-imaging system (Molecular Dynamics) and analysed usingImageQuant software.

Results:

Southern analysis of a BamH1 digest of genomic DNA extracted fromtransgenic Arabidopsis thaliana plants was carried out using as a probean 810 base pair 32P-labelled PCR fragment that was amplified from EST57400 with the primers 57400_A3 (5′ AGAGTTCACTTGCAAGCTGA 3′ SEQ ID NO:3)and 57400NR1 (5′ GGATGCTTCCCT 3′ (SEQ ID NO:4)). The size of the BamH1restriction fragment containing cDNA for alpha-farnesene synthase is2050 base pairs. The results are shown in FIG. 17. The lane labelled MWis the molecular weight marker (Invitrogen). pHEX is refers to genomicDNA extracted from transgenic Arabidopsis thaliana plants containing thetransformation vector only, without the alpha-farnesene synthase cDNAinsert. Lanes labelled 1, 2, 3 and 4 are independent transgenicArabidopsis thaliana lines containing the alpha-farnesene synthase cDNAinsert.

RT-PCR Amplification:

RT-PCR (Platinum® Quantitative RT-PCR Thermoscript One-Step System,Invitrogen) amplifications were performed according to themanufacturer's recommendations on total RNA extracted from Arabidopsisthaliana seedlings, leaves and flowers of Line 3. cDNA synthesis was at60° C. for 30 minutes, followed by denaturation at 96° C. for 5 minutes,then 40 cycles of amplification involving denaturation at 96° C. for 30seconds, annealing at 55° C. for 40 seconds and extension at 72° C. for60 seconds. For the final cycle extension at 72° C. was continued for afurther 5 minutes. Prior to RT-PCR the total RNA was treated with DNaseI (Life Technologies) for 10 minutes at room temperature. Concurrentlywith RT-PCR amplification, PCR amplification was also performed on theDNase I-treated total RNA to check for genomic DNA contamination. ThePCR primers were as follows: 57400_A3 AGAGTTCACTTGCAAGCTGA (SEQ ID NO:3)and 57400_A4 GAAAAGTTCCAGCATTCCTT (SEQ ID NO:8), 5′ amplification;57400NF1 GCACATTAGAG AACCACCAT (SEQ ID NO:9) and 57400NR1 GGATGCTTCCCT(SEQ ID NO:4), internal amplification; 57400_A1, CTTCACAAGAATGAAGATCT(SEQ ID NO:10) and 57400_A5 TTCCATGCATTGTCTATCAT (SEQ ID NO:11), 3′amplification. The resulting amplification products were analysed as forPCR amplification. The results confirmed the presence of RNA transcriptscorresponding to the transgene (see FIG. 18).

Arabidopsis Proof of function studies: Approximately 200 T2 generationseeds were measured into a microcentrifuge tube from each of fourindependent overexpressing lines and one control line. The seed wassterilised in a 1.5% bleach solution containing 0.01% Triton-X, andincubated for 15 min with occasional mixing. The seed was washed severaltimes with distilled water, and resuspended in 0.1% agarose, prior toplating on 0.5X MS media, containing 100 mg/mL kanamycin. The plateswere placed into growth rooms with a 12-hour light/12-hour dark cycle.After 2-3 weeks growth 30 plants from each line were transferred to soilin pots and were allowed to continue growing in the glasshouse untilmature and producing inflorescences.

Between 30 and 50 inflorescences were harvested before siliqueappearance and cut ends immediately placed in water. These were thentransferred into 55 mL glass tubes with a ground glass joint socketwhich contained 5 mL distilled water with 25 μM FDP and 7 mM MgCl₂. Thetest-tubes were packed tightly with plant material. The tube was sealedwith a ground glass inlet stopper containing a gas line and a volatilesorbent cartridge containing 100 mg Chromosorb 105. The headspace in theflask was purged with dry air at 50 ml min⁻¹ while being trapped for 55hours. The Chromosorb cartridge was dried with a N₂(g) flow at 10 psi,35 C for 15 min prior to analysis. The volatiles were thermally desorbedand analysed in the same manner as previously described for the apples.

Results: There were a number of sesquiterpene compounds found in theinflorescences of the Arabidopsis thaliana plants. The product E, Ealpha-farnesene was found in all 4 overexpressing lines of the plantstested and in control pHEX plants. This was confirmed by retention timeand comparison of the mass spectra with library spectra. In the 4 lineswhich had been transformed with EST57400, the ratio of thecaryophyllene: alpha-farnesene peak was 3:1, in the pHEX controlalpha-farnesene was also present but the ratio of the caryophyllene peakto the alpha-farnesene peak was greater than 10:1. (alpha-farnesenepeaks were at 43.33, and 43.41 minutes respectively for pHex, and line 3see FIG. 19). This suggests that the alpha-farnesene is being producedin small quantities in A. thaliana inflorescences along with the othersesquiterpenes but where we have added the gene, alpha-farnesene isbeing produced in greater proportions than in control plants.

Northern Analysis in Apple (‘Royal Gala’):

Methods:

Northern Blot Analysis

Northern analysis was performed as described by Rueger et al. (1996)using antisense RNA probes. Probe templates for alpha-farnesene synthasewas prepared by PCR (Genius thermocycler, Techne, Cambridge, UK) plasmidDNA using the following primers:

57400NF1 (SEQ ID NO:9) 5-GCACATTAGAGAACCACCAT-3 and 57400NR1 (SEQ IDNO:12) 5-TAATACGACTCACTATAGGGATGCTTCCCTTAAGTTTT-3Final reaction components were as follows: 1×Taq polymerase buffer(Invitrogen), 200 mM dNTPs, 1.5 mM MgCl2, 200 pM of each primer, 50 ngplasmid template or 25 ng genomic DNA, 1 unit Platinum® Taq DNApolymerase in a final volume of 50 mL. PCR conditions includeddenaturation at 94° C. for 4 min, followed by 25 cycles at 94° C. for 30seconds, 55° C. for 30 seconds and 72° C. 30 seconds.

Probe transcription reaction for alpha-farnesene synthase was preparedusing T7 RNA polymerase (Invitrogen) according to manufacturer'sinstructions with one modification. The reaction was supplemented with70 mM DIG-11-UTP (Roche), with UTP reduced to 130 mM. Transcriptionreactions were incubated at 37° C. for 1 h and then treated with 1 unitof RQ1 RNase free DNase (Promega) in 50 μL total volume for a further 15min at 37° C. The quantity of RNA probe was calculated by measuringabsorbance at 260 nm of a 5 mL aliquot diluted 1:80 in water. Theconcentration was halved owing to the effect of DIG, and used at therate of 100 ng probe per mL of hybridisation buffer. Equality of RNAloading was visualised through staining of RNA gels with ethidiumbromide, and after probing the blot with an 18S ribosomal RNA PCRproduct:

18S-RFT: CTGGCACCTTATGAGAAATC (SEQ ID NQ:13) 18S-RTR:CCACCCATAGAATCAAGAAA (SEQ ID NO:14)RT-PCR, 55° C. annealing giving a 343 bp product with 40% GC content for42° C. EasyHyb hybridization.

The level of alpha-farnesene synthase mRNA was adjusted for loadingdifferences calculated from hybridisation of the 18S ribosomal RNA. Theresulting signals were analysed using ImageQuant software and ahistogram of alpha-farnesene synthase mRNA levels was plotted.

Virtual Northern

ESTs in the HortResearch EST database that were related toalpha-farnesene synthase were identified by using the gene sequence in aBLAST NRDB90 search (Altschul et al., 1997). A ‘virtual northern’ of thetissues that the EST sequences were found in was produced from ananalysis of the cDNA libraries present in the database.

Results:

In a virtual northern, 1 EST was identified among 1000 ESTs in floralbuds, 2 from 8050 ESTS in ripe (150 DAFB) apple skin, and 1 with alonger 3′ UTR from apple cortex 126 DAFB (days after full bloom) fromamong 4,500 ESTs. Hence the gene is expressed at relatively low levelsduring fruit development.

ESTs were identified in mature leaves from three different cultivars,Aotea, Pinkie and senescing Royal Gala leaf. Sequence at the DNA levelfor all except the ESTs from Aotea and Pinkie were identical to thealpha-farnesene synthase cDNA sequence. No other homologous sequenceswere detected.

In a standard northern analysis, expression of alpha-farnesene synthasewas greatest in skin of ripe fruit (150DAFB) followed by expanding leaf(Aotea) (FIG. 20). The gene was expressed throughout fruit growth,although at lower levels than in ripe fruit skin. mRNA encodingalpha-farnesene synthase was either too low to be detected or notpresent in floral meristems, phloem and very young fruitlets.

Phylogenetic Analysis:

Computational analysis was performed using the European MolecularBiology Open Software Suite (EMBOSS) (Rice et al., 2000). Sequenceidentity and similarity was calculated using the pair wise alignmentprogram Needle, which uses the algorithm of Needleman and Wunsch (J.Mol. Biol. 48; 443-453 (1970). The default parameters were used (Gapextension penalty: 0.5 for any sequence; Gap opening penalty: 10 for anysequence). Sequence relatedness was analysed using CLUSTAL X and trimmedand shaded using the program GeneDoc Nicholas and Nicholas, 1997).Phylogenetic trees were used generated in CLUSTAL X using theneighbour-joining method, and the uprooted trees visualised usingTreedraw.

The full length alpha-farnesene synthase was compared to all otherterpene synthase sequences of known function (FIGS. 21 a and 21 b). Itformed a clade with a single member, well separated from the nearesthomologues, two isoprene synthases from poplar. The separation into itsown group reinforces the dissimilarity of the protein sequence to bothother sesquiterpene synthases and to monoterpene synthases. A similarresult was obtained when only the active site metal binding regionaround the DDxxD motif was compared across the same set of sequences. Inshort, this gene would not have been predicted to be a sesquiterpenesynthase.

The above Examples illustrate of practice of the invention. It will beappreciated by those skilled in the art that the invention can becarried out with numerous modifications and variations. For example,variations to the nucleotide sequences may be used and the sequences maybe expressed in different organisms.

1. An isolated polynucleotide having at least 90% sequence identity tothe sequence of SEQ ID NO:1 wherein said polynucleotide encodes apolypeptide with alpha-farnesene synthase activity.
 2. The isolatedpolynucleotide as claimed in claim 1 wherein the sequence has at least95% identity to the nucleotide sequence of SEQ ID NO:1.
 3. The isolatedpolynucleotide as claimed in claim 1 wherein the nucleotide sequence isthat of SEQ ID NO:1.
 4. An isolated polynucleotide encoding apolypeptide having at least 90% sequence identity to SEQ ID NO:2,wherein said polypeptide has alpha-farnesene synthase activity.
 5. Theisolated polynucleotide as claimed in claim 4 wherein the polypeptidehas at least 95% identity with the amino acid sequence of SEQ ID NO:2.6. The isolated polynucleotide as claimed in claim 4 wherein thepolypeptide has the sequence of SEQ ID NO:2.
 7. A genetic constructcomprising the polynucleotide of claim
 1. 8. A genetic constructcomprising in the 5′-3′ direction an open reading frame polynucleotideencoding the polypeptide of claim
 1. 9. The genetic construct as claimedin claim 8 further comprising a promoter sequence.
 10. The geneticconstruct as claimed in claim 9 which further comprises a terminationsequence.
 11. The genetic construct as claimed in claim 10 wherein thesequence of the encoded polypeptide has the amino acid sequence of SEQID NO:2 or a fragment thereof with alpha-farnesene activity.
 12. Avector comprising the genetic construct of claim
 7. 13. A host cellcomprising the genetic construct of claim
 7. 14. A transgenic plant cellwhich includes the genetic construct of claim
 7. 15. A transgenic plantcomprising the plant cell as claimed in claim 14.